The present invention relates to the controlled freezing of biological samples consisting of cells and tissues, such as semen, oocytes, and embryos; and, more particularly, to a directional freezing device that sets up a laterally varying temperature gradient, and freezing and thawing protocols allowed by the device.
When a biological sample containing living cells in a freezing solution is frozen, the first portion of the sample to freeze is the intercellular fluid. The formation of ice in the intercellular fluid increases the salt concentration there. If the sample is frozen too slowly, the high concentration of salt in the intercellular fluid may kill the cells, by osmotic shock or by chemical toxicity. Conversely, freezing the sample too rapidly may lead to the formation of intracellular ice crystals, which also kill the cell, by internal mechanical damage. In addition, the rate of cooling affects the morphology of the intercellular ice crystals. Morphologies such as closely packed needles also kill cells, by external mechanical damage. Thus, maximizing the survival rate of cells subjected to freezing and thawing requires careful control of the freezing process.
An alternative method of freezing biological samples, which totally avoids the problems associated with ice crystal formation, is to cool them so fast that the intercellular and intracellular fluids vitrify instead of crystallizing as ice. This method has dangers of its own, however. In particular, the rate of cooling is so fast that, because of thermal shock, glass fractures may form within the sample at temperatures below its glass transition temperature. To prevent ice crystal formation upon thawing, vitrified samples must be warmed as fast as they were cooled, so thermal shock may cause fracture formation either during the cooling process or during the warming process.
The conventional method for freezing biological samples is to place them in a chamber and lower the temperature of the chamber in a controlled manner. Samples frozen in this manner freeze from the outside in. The thermal gradient within the sample is determined implicitly by the temperature of the chamber and the thermal conductivities of the materials within the sample, and is not explicitly controllable. This makes it difficult to achieve the optimal cooling rate, which minimizes both the toxicity associated with cooling too slowly and the mechanical damage associated with cooling too fast.
Rubinsky, in U.S. Pat. No. 4,531,373, introduced controlled directional freezing, in which a sample is placed on a microscope slide, and the microscope slide is moved longitudinally through a region of substantially constant temperature gradient dT/dx (T denoting temperature and x denoting distance). If the microscope slide is moved through the temperature gradient at a constant speed V=dx/dt, where t denotes time, then each point in the sample cools at a rate of dT/dt=V*(dT/dx). Using Rubinsky's method, the rate of cooling of each point in the sample is subject to explicit control. In addition, if the cooling is done on a microscope stage, the sample can be monitored in detail for undesired phenomena such as the formation of intracellular ice.
Rubinsky's method, having only one uniform thermal gradient, is inherently limited to cooling at a single rate. Thus, it is unsuitable for cooling protocols that require different rates in different temperature ranges. For example, Arav ("Vitrification of oocytes and embryos", in Embryonic Development and Manipulation (Lavria and Gandalfi, editors), Portland Press, 1992, pp. 255-264) recommends that vitrification be done with rapid cooling above the glass transition temperature and slower cooling below the glass transition temperature. In addition, Rubinsky's use of a microscope stage for monitoring makes his device unsuitable for commercial or industrial scale production, or for the use of commercial cell packaging ("straws").
There is thus a widely recognized need for, and it would be highly advantageous to have, a device for directional cooling of a biological sample by moving the sample through regions of laterally varying temperature gradient, and associated freezing and thawing protocols that exploit the ability to cool and thaw at different rates in different temperature ranges.